PROJECT SUMMARY During type 2 diabetes (T2D), loss of insulin sensitivity strongly associated with hepatic lipid accumulation increases gluconeogenesis underlying chronic hyperglycemia. Furthermore, increased hepatic de novo lipogenesis (DNL) during T2D is thought to drive insulin resistance and non-alcoholic fatty liver disease (NAFLD). Thus, identifying mechanisms directly modulating both hepatic DNL and gluconeogenesis could be uniquely valuable for understanding T2D pathophysiology and therapeutic opportunity. Cytosolic citrate is believed to be a master regulator of hepatic metabolism by reciprocally regulating glycolysis and gluconeogenesis and by supplying substrate and reducing power for DNL. Citrate is produced in the mitochondria and requires a specific transporter, the mitochondrial citrate carrier (CiC), to reach the cytosol. Thus, the CiC is predicted to occupy a central metabolic node linking hepatic mitochondrial metabolism, DNL, glucose, and reductive drive. Yet, surprisingly, the role of the CiC modulating hepatic DNL and gluconeogenesis in normal and T2D states remains sparsely addressed in vivo. The overall goal of this application is to understand how the hepatic CiC contributes to fundamental metabolism and T2D pathophysiology. This will be addressed by pursuing two specific aims: 1) Determine how hepatic CiC function regulates hepatic DNL in T2D states; and 2) Determine how hepatic CiC function contributes to hyperglycemia in T2D states. Experiments in aim 1 will test the hypothesis that disrupting hepatic CiC activity in vivo during T2D states decreases DNL, by decreasing supply of citrate as a carbon source and decreasing NAPDH available for fatty chain elongation. Experiments in aim 2 will test the hypothesis that disrupting hepatic CiC activity in vivo during T2D states decreases hyperglycemia by attenuating liver insulin resistance, shifting hepatic glucose metabolism towards glycolysis away from gluconeogenesis, and decreasing mouse correlates of NAFLD progression. Overall, the proposed investigation will test the fundamental regulatory role of the CiC in vivo and provide novel, mechanistic information on how the single metabolic step of mitochondrial citrate export contributes to the core T2D features of increased hepatic DNL and gluconeogenesis. This research is significant because successful completion will uniquely advance fundamental understanding of T2D pathophysiology. This research is innovative because it will utilize novel in vivo CiC disruption and metabolomic tracing models to test the role of the CiC role linking mitochondrial metabolism, DNL, and gluconeogenesis in T2D.